Gut Microbiota-mediated Alleviation of Dextran Sulfate Sodium-induced Colitis in Mice

Background and Aims Gut dysbiosis characterized by an imbalanced microbiota is closely involved in the pathogenesis of a widespread gastrointestinal inflammatory disorder, inflammatory bowel disease. However, it is unclear how the complex intestinal microbiota affects development or resistant of mucosal inflammation. Our aim was to investigate the impact of the gut microbiota on susceptibility in a mouse model of ulcerative colitis. Methods We compared the susceptibility to dextran sulfate sodium (DSS)-induced colitis of inbred BALB/c mice obtained from the 3 main distributors of laboratory animals in Japan. Clinical symptoms of the colitis and the faecal microbiota were assessed. Cohousing approach was used to identify whether the gut microbiota is a primary factor determining disease susceptibility. Results Here, we showed differences in the susceptibility of BALB/c mice from the vendors to DSS colitis. Analysis of the gut microbiota using 16S ribosomal RNA sequencing revealed clear separation of the gut microbial composition among mice from the vendors. Notably, the abundance of the phylum Actinobacteriota was strongly associated with disease activity. We also observed the expansion of butyrate-producing Roseburia species in mice with decreased susceptibility of the disease. Further cohousing experiments showed that variation in clinical outcomes was more correlated with the gut microbiota than genetic variants among substrains from different suppliers. Conclusion A BALB/c substrain that was resistant to DSS-induced colitis was observed, and the severity of DSS-induced colitis was mainly influenced by the gut microbiota. Targeting butyrate-producing bacteria could have therapeutic potential for ulcerative colitis.


Introduction
L aboratory mice are important species for preclinical animal experiments in biomedical research and contribute to mechanistic studies and drug development in the context of various human diseases.Inbred mouse strains difference can be a reason of host immune characteristics as well as behavioural phenotypes. 1,2Numerous substrains have been derived from original inbred strains. 3Substrains are defined as branches of an inbred strain produced by separated brothersister mating over at least 20 generations from multiple vendors.4][15] Inflammatory bowel disease (IBD) is a multifactorial immunemediated inflammatory disease that includes 2 conditions, Crohn's disease and ulcerative colitis. 16,179][20][21] SCFAs produced by gut bacteria act as coenzymes in fat and carbohydrate metabolism, thus exhibiting anti-inflammatory effects in IBD.Among the SCFAs, butyrate serves as a principal energy source for intestinal wound healing and barrier function.The gut microbiota of IBD patients exhibits a selective decrease in the levels of butyrate producers, and the colonocytes of IBD patients are incapable of transferring and utilizing butyrate. 22he dextran sulfate sodium (DSS)-induced colitis model is routinely used as a principal mouse model of ulcerative colitis.DSS administration in drinking water mediates gut epithelial damage, which causes inflammation.Mouse strain and sex differences influence colitis susceptibility. 23For example, similar to the clinical development of ulcerative colitis in humans, male mice are more likely to develop DSSinduced colitis than female mice. 24,25Additionally, BALB/c mice require higher concentrations of DSS to induce colitis than C57BL/6J mice. 24C57BL/6 wild-type mice from the same inbred strain purchased from 2 vendors (substrains), Jackson laboratory and Taconic farms, showed different bacterial compositions, and Jackson mice showed significantly fewer species of bacteria. 26Colonization of the gut by a segmented filamentous bacterium was found only in Taconic mice, and the bacterium induced the production of inflammatory Th17 cells in the lamina propria of the small intestine, which resulted in increased resistance to Citrobacter rodentium-induced colitis. 26In addition, the composition of gut microbes that influence susceptibility to several diseases, including abdominal sepsis, vary among substrains. 27espite these advances in understanding, whether the gut microbiota is a primary factor determining disease susceptibility in DSS-induced colitis remains unknown.We compared mice from the same inbred strain (BALB/c) that were obtained from the 3 main distributors of laboratory animals in Japan and identified considerable variability in the presentation of DSS-induced colitis among mice from different vendors.We, therefore, quantified the influence of the faecal microbiota associated with mice from different vendors.We identified that mice from each vendor harboured a distinct gut microbiota.Thus, using a cohousing approach, we revealed that disease resistance mainly relies on the gut microbiota and not on genetic differences among substrains.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (Protocol number: A2021-088C9) and the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (R05-009-0).All animals that were used in this study were housed in groups of 3-6 mice, fed standard pellet diet, under a 12-hour light/dark cycle.

16S rRNA Sequencing
Mouse faeces or colon luminal contents were collected on day 0 and day 8. Collected samples were stored at À80 C until further use.Bacterial DNA was isolated using a NucleoSpin DNA stool kit (Takara Bio, Shiga, Japan).The V3-V4 regions of the 16S ribosomal RNA (rRNA) gene were amplified in each sample.Sequencing was performed on the Illumina MiSeq platform using a MiSeq Reagent Kit V3 (300 bp x 2) (Eurofins genomics, Tokyo, Japan and Bioengineering Lab.Co., Ltd., Kanagawa, Japan).
Raw sequences were curated using the software package Qiime2.Sequences were assigned to operational taxonomic units using a cut-off ¼ 0.03 and classified using the SILVA platform with a 70% confidence threshold.We used the linear discriminant analysis effect size (LEfSe) method 28 (http://huttenhower.sph.harvard.edu/lefse),which is used to perform a combined assessment of statistical significance and biological relevance.

Cohousing
For the cohousing experiment, four-week-old BALB/C female mice purchased from SLC and Charles River were housed separately for one week in the same room and were fed the same diet before cohousing.Then, the mice were transferred into a new cage, and SLC mice and Charles River mice were cohoused for 4 weeks as described previously. 29SLC mice or Charles River mice that were kept in separate cages were used as controls.Acute colitis was induced with 4% (w/v) DSS for 7 days afterwards.

Statistics and Reproducibility
Comparisons of 2 groups were performed using an unpaired t (parametric) test or a Mann-Whitney U (nonparametric) test.Differences among more than 3 groups were evaluated using oneway analysis of variance for parametric analysis or the Kruskal-Wallis test for nonparametric analysis followed by Bonferroni correction (parametric) or Steel-Dwass correction (nonparametric).The normality of the data was analyzed using the Kolmogorov-Smirnov test.Homogeneity of variance was analyzed using the F test (2 groups) or Bartlett test (more than 3 groups).The sample distribution of the gut microbiota was analyzed by a non-metric multi-dimensional scaling method.Correlation analysis was performed using the Pearson correlation coefficient.Error bars represent the standard deviation of a data set.All statistical analyses were performed with R statistical software.

Results
The Phenotype of DSS-Induced Colitis Varied Among Mice From Different Vendors We compared female six-to seven-week-old BALB/C wild-type mice obtained from 3 vendors: SLC, CLEA, and Charles River.Acute colitis was induced by the administration of 4% (w/v) DSS in drinking water for 8 days.To investigate whether colitis induction by DSS was comparable among mice from different vendors, weight loss, rectal bleeding, stool consistency, colon shortening, and spleen enlargement were observed as clinical features of disease.Regarding weight loss, the body weights of mice from all vendors slightly increased during the first few days of the experiment.The weight of CLEA mice and Charles River mice gradually began to decrease afterward, and the body weights on day 8 were 14.4 AE 9.7 and 16.8 AE 5.7% below the initial weight, respectively (Figure 1A).SLC mice showed little weight loss, and the body weight on day 8 was only 1.5 AE 4.1% below the initial weight.The level of weight loss in CLEA mice and Charles River mice significantly differed from that in SLC mice on day 7 (P ¼ .04 and .03,respectively) and day 8 (P ¼ 8.0 Â 10 -3 and 4.0 Â 10 -3 , respectively).Similarly, the DAI score, which comprises the degrees of weight loss and intestinal bleeding, was significantly higher (indicating more severe colitis) in CLEA and Charles River mice than in SLC mice on day 7 (P ¼ .04 and .02,respectively) and day 8 (P ¼ .02and .02,respectively) (Figure 1B).The DAI score ranges from 0 to 12 (total score).Moreover, SLC mice did not exhibit considerable colon shortening (Figure 1C and D) or spleen enlargement (Figure 1E), unlike CLEA or Charles River mice.There was a trend of more colitis symptoms in Charles River mice than in CLEA mice, although significant differences in these symptoms of colitis were not observed.

The Gut Microbial Compositions of BALB/c Mice Largely Varied Among Mice From Different Vendors
We compared the gut bacterial composition of mice from SLC, CLEA, and Charles River using 16S rRNA sequencing.Nonmetric multidimensional scaling ordination using Horn-Morisita dissimilarities based on community membership indicated a clear separation of the microbiota among mice from the different vendors before DSS treatment (Figure 2A).Consistent with previous studies, a significant decrease in microbial diversity, which is a characteristic of dysbiosis, was observed in CLEA and Charles River mice, whereas SLC mice did not show diversity changes after DSS administration (Figure 2B).Microbial diversity was not significantly different among vendors either before or after DSS administration.Fifteen phyla were identified in total, as shown in Figure 2C.Contrary to our expectations, Charles River mice, but not SLC mice, were distinct from mice from other vendors before DSS treatment.In other words, SLC mice and CLEA mice were similar despite differences in disease severity.Notably, although Bacteroidetes and Firmicutes are 2 main phyla of the gut microbiota, the phylum Firmicutes was the most dominant in Charles River mice before DSS treatment, comprising up to 95%; hence, a low abundance of Bacteroidetes (2.2%) was observed.The phylum Firmicutes consists of the class Bacilli and class Clostridia (Figure A1), and the levels of Clostridia in mice before DSS treatment was significantly lower in mice from SLC compared to CLEA and Charles River (Figure 2D), suggesting that the high Firmicutes abundance in Charles River mice was mainly due to the class Clostridia.The levels of Clostridia in mice after DSS treatment was not significantly different among vendors.Further investigation of the taxa comprising the class Clostridia was performed, and the levels of abundant families that were !1% abundant in at least one group are shown in Figure 2E.A decrease in the levels of SCFA-producing Clostridium cluster IV (Ruminococcaceae and Clostridia UCG-014) and XIVa (Lachnospiraceae) is often associated with gut dysbiosis. 30The levels of Lachnospiraceae and Rumicococcaceae in mice before DSS treatment were significantly lower in mice from SLC compared to CLEA and Charles River.The level of Oscillospiraceae in mice before DSS treatment was significantly lower in mice from SLC compared to Charles River.In addition to the presence of 2 main phyla, Firmicutes and Bacteroidetes, the analysis of the microbiota before DSS treatment showed a very strong association between the DAI inflammation score and Actinobacteriota proportion (R 2 ¼ 0.80), although the abundance of this microbe was quite low (Figure 2F and Figure A2).To distinguish the bacterial taxa that are commonly found in mice from each vendor, differences in microbial taxa at the family level among vendors were calculated by LEfSe as well as a heatmap (Figure 3 and Figure A3).Eight taxa out of 13 that were significantly abundant in Charles River mice before DSS treatment were from the class Clostridia except Lactobacillaceae, RF39, Deferribacteraceae, Erysipelatoclostridiaceae, Acholeplasmataceae, and Eggerthellaceae.The family Eggerthellaceae is a member of phylum Actinobacteriota, and all Actinobacteriota bacteria found in this study belonged to the Eggerthellaceae.Contrary to the mice before DSS treatment, taxa comprising the class Clostridia were significantly lower levels in Charles Rimer mice after DSS treatment.Members of the family Desulfovibrionaceae and Rikenllaceae were significantly abundant in SLC mice both before and after DSS treatment.More detailed composition at genus level by LEfSe analysis indicated similar tendency (Figure 4A and B).Members of the genus Desulfovibrio and Bilophila, both comprising family Desulfovibrionaceae, were significantly abundant in SLC mice both before and after DSS treatment.Twenty taxa out of 28 that were significantly abundant in Charles River mice before DSS treatment were from the class Clostridia and there were no abundant taxa in mice after DSS treatment.Furthermore, genus Roseburia is an only taxon comprising the class Clastridia in SLC mice after DSS treatment (Figure 4B).Among the SCFAs producing bacteria, Roseburia intestinalis (a member of family Lachnospiraceae) and Faecalibacterium prausnitzii (a member of family Oscillospiraceae) are the primary butyrate producers in the human gut. 31The mean abundance of the genus Roseburia was higher in Charles River and CLEA mice than in SLC mice before DSS treatment (3.1%, 0.81%, and 0.05%, respectively), and the abundance decreased in Charles River and CLEA mice after DSS treatment, whereas that in SLC mice increased after DSS treatment (0.17, 6.4 Â 10 -3 , and 0.68%, respectively) (Figure 4C).The genus Faecalibacterium was not identified in all samples.

Commensal Intestinal Bacteria From Mice With Severe Colitis Influence the Severity of Disease in Mice With Milder Colitis
Our gut microbiota analysis suggested that specific colonic microbes may induce colitis susceptibility.We next aimed to address the possibility that disease resistance to colitis in SLC mice might have been due to genetic variants among BALB/c mice substrains from the different vendors.Therefore, we cohoused SLC mice (mildest colitis symptoms) with Charles River mice (severest colitis symptoms) to allow horizontal bacterial transmission.SLC mice that were cohoused with Charles River mice for 4 weeks showed statistically higher DAI scores than SLC mice that were kept in separate cages (day 7, P ¼ .03),suggesting that the gut microbiota, rather than the presence of genetic variants, is a dominant factor in the variable response to DSS between these 2 mouse substrains (Figure 5A).Regarding Charles River mice, although both Charles River mice housed separately and cohoused mice showed significantly higher DAI values than SLC mice (day 7, P ¼ .03and .01,respectively), no change was observed due to cohousing with SLC mice.Similarly, more colon shortening was observed in cohoused SLC mice, separately housed Charles River mice, and cohoused Charles River mice than in separately housed SLC mice (P ¼ 1.6 Â 10 -4 , 2.0 Â 10 -3 , and 1.7 Â 10 -3 , respectively) (Figure 5B and C).We further analyzed the gut microbiota of cohoused SLC mice using 16S rRNA sequencing and compared bacterial compositions to those from solo housed SLC, CLEA, and Charles River mice.After 4 weeks of cohousing, SLC mice cohoused with Charles River mice maintained the Firmicutes/Bacteroidota balance and the bacterial compositions were generally similar to solo housing SLC (Figure 6A and B).Regarding the butyrateproducing genus Roseburia, the abundance of Roseburia in cohoused SLC mice was 0.38% after cohousing and decreased to 0.19% with DSS challenges.The decrease in Roseburia in cohoused SLC mice indicates loss of persistence of Roseburia, unlike disease-resistant solo housed SLC mice (Figures 4C and 6C).Furthermore, we performed LEfSe analysis to compare disease-resistant SLC mice with disease-susceptible Charles River, CLEA, and cohoused SLC mice following DSS treatment.The 6 genera were abundant in disease-resistant SLC mice, Turicibater, Roseburia, Alistipes, Rikenellaceae RC9 gut group, Ruminococcaceae, and Parasutterella (Figure 6D).

Discussion
The DSS-induced colitis model is widely used because it can be established quickly and is simple. 24Here, we found clear differences in susceptibility to DSS-induced colitis among Japanese laboratory mice from 3 vendors.BALB/C mice purchased from SLC showed lower disease symptoms than mice from the other 2 vendors.The gut microbiota is known as an indispensable factor in gut inflammation. 32To quantify the connection between disease severity and the gut microbiota, the faeces of mice from 3 vendors collected before and after DSS treatment were compared using 16S rRNA sequencing, revealing that each group of mice from the different vendors harboured a distinct gut microbiota.Moreover, the cohousing data from the present study further show that the severity of DSS-induced colitis was mainly influenced by the gut microbiota.
The variability in DSS-induced colitis among individual mice from the same inbred strain has been documented in a large-scale animal experiment using genetically identical laboratory mice from a single animal facility. 33The researchers reported that the presence of specific gut bacteria was mainly responsible for the variable experimental outcomes in the DSS model.In humans, a large clinical cohort study examined genetic-microbial associations in healthy people who had different ancestral backgrounds but shared a relatively similar environment.The study showed that host genetics or ancestral backgrounds have a minor role in determining the gut microbiome; rather, the microbiota is shaped predominantly by lifestyle and is similar among individuals who share a relatively homogenous environment. 34IBD patients show a distinct distribution of certain bacterial taxa; IBD is accompanied by a decreased abundance of Bacteroidetes, Firmicutes, Clostridia, Lactobacillus, and Ruminococcaceae and an increased abundance of Gammaproteobacteria and Enterobacteriaceae. 35,36In particular, the Firmicutes/Bacteroidetes ratio is widely accepted to have vast influences on the maintenance of gut homeostasis, and an imbalance in these taxa can lead to various pathologies. 32Related to these epidemiological studies, our study revealed a higher percentage of Firmicutes in Charles River mice, and these bacteria were classified mainly into the class Clostridia.8][39] In particular, Roseburia intestinalis is one of the primary butyrate producers in the human gut. 31The genus Roseburia consists of obligate gram-positive anaerobic bacteria, all of which are known to be SCFA producers. 40We reported that the abundance of the genus Roseburia increased precipitously in SLC mice after DSS treatment, whereas cohoused SLC mice as well as mice from other 2 vendors decreased the abundance of Roseburia along with DSS treatment.This implies that the persistence of Roseburia correlates with decreased susceptibility to disease in this setting.In addition to Roseburia, 5 genera were more abundant in disease-resistant SLC mice than in cohoused SLC mice and mice from other 2 vendors shown in Figure 6D.Some members of genera Turicibater, Alistipes, and Ruminococcaceae were also known as SCFA-producers. 41Similarly, in a previous study that compared the gut microbiota of mice from 2 vendors (substrains) in the United States, colonization of the gut by colitis-resistant Candidatus arthromitus (a segmented filamentous bacterium and member of the family Clostridiaceae) was found in Taconic mice. 26The distribution of disease-resistant bacteria may not be ubiquitous but may have similar characteristics among different mice.
As previously discussed, we showed that few colitis symptoms were observed in SLC mice in our study.Although SLC is a major animal manufacturer, there have been few reports of studies using mice from SLC in the field of DSSinduced colitis research.One report using male C57BL/6 mice from SLC described body weight loss as well as a low DAI score, which supports our data. 42C57BL/6, BALB/c, and C3H/ HeJ strains are known to be genetically susceptible to DSSinduced colitis. 43To our knowledge, this is the first report to describe a disease-resistant BALB/c substrain with lower disease susceptibility, and we showed that the susceptibility  mainly relies on the gut microbiota.5][46] Considering these findings, members of the family Muribaculaceae could also be studied in the future as potential protective bacteria against disease.
In summary, a mouse substrain that was resistant to DSSinduced colitis was observed, and the severity of DSS-induced colitis was mainly influenced by the gut microbiota.DSSinduced colitis is one of the central preclinical models used in the gastrointestinal field.When studying disease susceptibility in laboratory mice, the mouse vendor and/or bleeding conditions that influence the gut commensal microbiota may be the reason for variable outcomes.

Figure 1 .
Figure 1.Clinical symptoms of colitis are highly variable among mice from different vendors.Mice from 3 commercial vendors, SLC, CLEA, and Charles River (CHA), were treated with 4% DSS for 8 days.Mice were evaluated daily, and weight loss and disease activity index scores were recorded.(A) Body weight changes, (n ¼ 5).(B) DAI score, a score from 0 to 12.A higher number indicates more severe colitis (n ¼ 5).(C) Gross images of the colon on days 0 (pre-DSS) and 8 (post-DSS).(D) Colon length was measured on day 8. (E) Gross images of the spleen on days 0 and 8. *P < .05,**P < .01.

Figure 2 .
Figure 2. The gut microbiota in mice from 3 vendors assessed using 16S rRNA sequencing.The colorectal microbial composition in SLC, CLEA, and Charles River (CHA) mice treated with 4% DSS for 8 days was assessed using 16S rRNA amplicon sequencing.n ¼ 4-5.(A) A nonmetric multidimensional scaling analysis identified a clear difference among mice vendor SLC (pink), CLEA (grey), and CHA (blue) before DSS treatment.(B) Dot plots show species-level microbial diversity measured by the Shannon diversity index.(C) Relative abundance of bacterial phyla presents in faeces on days 0 (pre-DSS) and 8 (post-DSS).(D) Relative abundance of class Clostridia comprising the phylum Firmicutes in faeces on days 0 (pre-DSS) and 8 (post-DSS).(E) Relative abundance of the bacterial family comprising the class Clostridia that was !1% abundant in at least one group of mice before DSS treatment.Significantly different among vendors indicates.(F) Relationship between DAI score and relative abundance of phyla Actinobacteriota in mice from vendor SLC (pink) CLEA (grey), and CHA (blue) before DSS treatment (R 2 ¼ 0.83).*P < .05,**P < .01.

Figure 3 .
Figure 3. Differences in the microbiota of mice from the 3 vendors at the family level.Differences in microbiota taxa at the family level among mice from the 3 vendors were calculated by LDA effect size (LEfSe) on day 0 (A) and day 8 (B) (n ¼ 4-5).Bacterial taxa comprising the class Clostridia.(C) Heatmap showing bacterial family frequency distribution across mice from the 3 vendors before DSS treatment (n ¼ 3).

Figure 4 .
Figure 4. Differences in the microbiota of mice from the 3 vendors at the genus level.Differences in microbiota taxa at the genus level among mice from the 3 vendors were calculated by LDA effect size (LEfSe) on day 0 (A) and day 8 (B).Bacterial taxa comprising the class Clostridia.(C) Relative abundance of the butyrate-producing genus Roseburia of mice before and after DSS treatment.

Figure 5 .
Figure 5. SLC mice cohoused with Charles River mice developed DSS-induced colitis.SLC mice (CO-SLC) and Charles River mice (CO-CHA) were cohoused for 4 weeks followed by 7 days of DSS administration (n ¼ 8).SLC mice (SLC) or Charles River mice (CHA) that were kept in separate cages were used as controls (n ¼ 6).Two independent experiments with identical results were combined.(A) DAI score.(B and C) Colon length was measured on day 7. *P < .05,**P < .01.

Figure 6 .
Figure 6.Comparison of gut microbiota from SLC mice cohoused with Charles River mice to solo housed mice from 3 vendors.The gut microbial composition in cohoused SLC mice (CO-SLC) was assessed using 16S rRNA amplicon sequencing and compared those from solo housed 3 vendors mice, SLC, CLEA, and Charles River (CHA).n ¼ 4-5.(A) Relative abundance of bacterial phyla presents in faeces before and after DSS treatment.(B) A nonmetric multidimensional scaling analysis of gut microbial compositions among mice vendor CO-SLC (green), SLC (pink), and CHA (blue) before (circle) and after (square) DSS treatment.(C) Relative abundance of the butyrate-producing genus Roseburia of CO-SLC mice before and after DSS treatment.(D) Differences in microbiota taxa at the genus level among mice from the 3 vendors as well as CO-SLC mice were calculated by LDA effect size (LEfSe) after DSS treatment.